One third of the American adult population is currently considered obese with a greater risk of developing cardiovascular disease and diabetes. While much work has focused on studying the genetic and dietary factors of obesity, less is known at the cellular level. In particular, how lipids and proteins interact at the surface of lipid droplets to promote lipid storage remains unclear. Our long range objective is to identify the role of proteins and lipids in regulating lipid droplet storage. The current proposal focuses on the role of one protein, adipose differentiation-related protein (ADRP), in regulating lipid storage through interactions with specific lipid domains and/or proteins at the surface of lipid droplets. Our specific aims are: (1) Identify and characterize lipid domains within lipid droplets. The working hypothesis is that the lipid droplet monolayer contains not only phospholipids, but also cholesterol and sphingolipids that spontaneously organize into raft- like microdomains, similar to those found in the plasma membrane. We will use techniques established by our laboratory to isolate and characterize lipid domains in lipid droplets. (2) Characterize protein-lipid interactions at the surface of lipid droplets. The working hypothesis is that select proteins associated with lipid droplets (including ADRP) play a key role in promoting lipid storage through interactions with specific lipids and/or proteins at the surface of lipid droplets. To date, we have shown that ADRP binds and colocalizes with cholesterol and fatty acids, yet no direct molecular interaction between lipids or other proteins has been shown. We propose to establish whether ADRP interacts with lipids and proteins at the surface of the lipid droplet using live cell FRET imaging to identify direct interactions. (3) Identify lipid binding motifs within lipid droplet associated proteins. We will test the working hypothesis that lipid droplet proteins (including ADRP) contain lipid binding motifs that interact with specific lipids by screening deletion mutants with lipid binding assays, CD analysis, and live cell FRET imaging to confirm protein-lipid interactions. In summary, we propose to study the role of lipid domains in organizing proteins and lipids within lipid droplets as a mechanism for lipid storage. The relevance of this work to public health will be in determining the mechanism by which lipids are stored to allow future development of therapeutic strategies to control and treat diseases related to aberrant lipid metabolism including cardiovascular disease, diabetes, and obesity.